PROJECT SUMMARY Sighs are critical for survival. They maintain normal lung functions by preventing atelectasis. Moreover, sighs have also been implicated in arousal, and failure to sigh has been associated with Sudden Infant Death Syndrome. The Ramirez lab has demonstrated that sighs are generated within the preBtzinger complex in the ventrolateral medulla. Indeed, the same neurons that generate eupneic inspiratory rhythmic activity seem to generate also sighs. This leads to an important, yet unresolved puzzle: How can the same neuronal network generate two types of breathing rhythm with strikingly different timing characteristics? The eupneic inspiratory rhythm occurs at a frequency of 1-2 Hz, while sighs are generated in the range of several minutes. Here we test the hypothesis that sighs are generated by neuroglial interactions that involve purinergic and glutamatergic interactions between glia and neurons with specific intrinsic and synaptic properties. This hypothesis is tested in three specific aims: Aim 1 will characterize the glial properties critical for the generation of sighs. We will evaluate the pharmacological, and neuromodulatory properties of isolated glia, and glia embedded within the respiratory network, using calcium imaging and optogenetic tools. Aim 2 will characterize the specific neuronal properties that are critical for the generation of sighs. We will test the hypothesis that these neuronal properties involve P/Q-type calcium channel dependent synaptic transmission that is modulated by metabotropic glutamate receptors (mGluR8), as well as intrinsic membrane properties that involve the persistent sodium current that is modulated by muscarinic receptors and beta-adrenergic receptors. Aims 1 and 2 will be performed in in vitro slice preparations that are amenable to a rigorous cellular level analysis. These two aims are complemented by aim 3 in which we will implement our insights gained in vitro in an in vivo preparation that is also amenable to optogenetic manipulations. This grant will provide critical insights into the generation of the sigh. In addition, this project will provide fundamental insights into neuroglial interactions. These insights will not only be relevant in the context of sigh rhythmogenesis, but these studies will also be important for understanding the generation of rhythmic behaviors in general.